Erythromycin Improves Glucocorticoid Resistance Caused By NETs Stimulation

Part I Erythromycin reduces monocyte glucocorticoid resistance induced by NETs stimulation is related to PI3K/Akt/mTor pathway genesObjective: 1.To study whether neutrophil extracellular traps(NETs)can cause glucocorticoid resistance in patients with chronic obstructive pulmonary diseases(COPD),and whether erythromycin(EM)can reduce Corticosteroid resistance caused by NETs.2.Transcriptomics was used to observe the effects of nets(neutral extracellular traps)on U937 cells’ genes at the genome-wide level,and use bioinformatics technology to discover genes related to glucocorticoid resistance.3.To study the related targets of regulating inflammatory response induced by nets at the genome-wide level,the signaling pathway and targets of EM regulating glucocorticoid resistance induced by nets were researched by bioinformatics technology.Methods: 1.Neutrophils were extracted from the peripheral blood of patients with chronic obstructive pulmonary diseases(COPD),and then Cigarette smoke extract(CSE)was used to induce neutrophils to produce NETs.NETs were used to stimulate the mononuclear cells extracted from the respective groups,and immunoblotting(Westernblot,WB)was used to detect whether there was glucocorticoid resistance after being stimulated by NETs.2.Then we studied the effect of NETs on the human lymphoma monocytic cell line U937 cells and the effect of EM on U937 cell induced by NETs:experimental group: 1.control group(Control);2.NETs group(NETs): 50ng/ml concentration NETs stimulated U937 cells for 24 hours;3.Erythromycin treatment group(EM+NETs): 1ug/ml concentration of erythromycin was pre-incubated for 24 hours,and 50ng/ml concentration of NETs was used to stimulate U937 cells for 24 hours.After the modeling expires,WB was used to detect histone deacetylase 2(HDAC2)and glucocorticoid receptor(GR)protein expression in each group.And Trizol cell lysate was used to lyse cell of ecah group,perform cell transcriptomics testing on the machine.After obtaining the transcriptome results,use the Limma package and WGCNA package of the R language and GSEA software to perform follow-up biometric analysis,and find genes or signal pathways related to inflammation,especially glucocorticoid resistance.Results: 1.NETs stimulation in COPD patients can increase the expression of Akt protein in peripheral blood mononuclear cells,and decrease the expression of HDAC2 and GR protein,suggesting the existence of glucocorticoid resistance.In in vitro experiments,NETs stimulation caused a decrease in the expression of HDAC2 and GR protein in U937 monocytes and inhibited GR phosphorylation.Erythromycin in U937 monocytes can reduce the decline of HDAC2 and GR protein expression caused by NETs stimulation,and can increase the phosphorylation level of GR.2.Compared with control cells,NETs stimulation at a concentration of 50ng/ml can significantly change the transcriptome of U937 cells and activate multiple inflammation-related genes.Erythromycin treatment can reverse part of the m RNA expression affected by NETs.Combined with the results of WGCNA and the screening of hub genes in the GSEA and PPI networks,the results showed that erythromycin mainly regulates inflammation and improves the glucocorticoid resistance induced by NETs was mainly through the following pathways: PI3K-δ/Akt/mTor pathway,IL-6/STAT3,NF-κB signaling pathway,p53,AP-1.Conclusions: 1.Nets produced in peripheral blood neutrophils of COPD patients can activate Akt in mononuclear cells,and inhibit the downstream HDAC2 and GR activities,which verifies that the stimulation of nets is related to glucocorticoid resistance in COPD.Erythromycin can reduce the inhibition of HDAC2 and GR expression caused by NETs stimulation,and can increase the phosphorylation level of GR and improve glucocorticoid resistance.2.NETs stimulation can cause inflammation and glucocorticoid resistance in U937 cells.The mechanism is mainly through the regulation of PI3K-δ/Akt/mTor pathway,IL-6/STAT3,NF-κB signaling pathway,p53,AP-1.3.Erythromycin can affect the inflammatory response of U937 cells caused by NETs stimulation via regulating multiple targets.The pathways related to the inflammatory response and glucocorticoid resistance caused by NETs are PI3K-δ/Akt/mTor pathway,IL-6 /STAT3,NF-κB signaling pathway,p53,AP-1.Among these pathways,the PI3K-δ/Akt/mTor pathway is the most relevant to glucocorticoid resistance.Part II Erythromycin reduces monocyte glucocorticoid resistance induced by NETs is correlated with PI3K/Akt/mTor pathway related proteinsObjective: 1.Use proteomics technology to explore(1)glucocorticoid resistance-related proteins and pathways caused by NETs and the influence of erythromycin on these proteins or pathways.(2)Combine proteomics and transcriptomics to study the effect of NETs on the inflammatory target m RNA and target protein in U937 cells,and observe the effect of erythromycin on the inflammatory target.2.Screen the proteins directly related to HDAC2 by Co-IP(Co-immunoprecipitation,Co-IP)combined with mass spectrometry,and verify the related proteins obtained by proteomics analysis.Methods: 1.Proteomics: Cell grouping: 1.Control group(Control);2.NETs group(NETs): 50ng/ml NETs stimulate U937 cells for 24 hours;3.Erythromycin treatment group(EM+NETs): After pre-incubating the cells with erythromycin(1ug/ml)for 24 hours,NETs(50ng/ml)stimulate U937 cells for 24 hours,then lyse the cells and take them to mass spectrometer for proteomics testing.2.Bioinformatics analysis: Use the String database and Cytoscape software to analyze the mass spectrometry results after Co-IP,establish a PPI network,and screen the key proteins.Use Blast2 GO Command Line,GSEA,KOALA(KEGG Orthology And Links Annotation)software,and Interpro database to annotate and analyze differential proteins.Through Fisher?s Exact Test,compare the distribution of each category,pathway,and target protein set in the overall protein set to evaluate the level of enrichment of related GO term,KEGG,and GSEA pathway proteins or functional domains.And jointly analyze the results of transcriptomics and proteomics to find out whether there is a common pathway.3.Co-IP combined mass spectrometry analysis: use RIPA cells to lyse U937 cells to obtain cellular protein,apply HDAC2 antibody for Co-IP,and take the precipitated protein to mass spectrometry for detection.4.Use the String database combined with Cytoscape software to analyze the mass spectrometric analysis results after Co-IP,and screen the proteins most closely related to HDAC2,and combine the mass spectrometric analysis results after Co-IP with the differentially expressed proteins obtained from proteomics analysis.Verify whether the differentially expressed protein is related to the HDAC2 protein.Results: 1.GO enrichment showed that NETs stimulation was related to the defense response of Gram-positive bacteria and negative bacteria,lipopolysaccharide binding,and cell response to lipopolysaccharide.At the same time,GO enrichment also showed that after the intervention of erythromycin,the differential protein is related to the positive regulation process of C-X-C chemokine binding,neutrophil degranulation,and IL-1β secretion.KEGG enrichment analysis showed that erythromycin regulates NETs-related reactive oxygen generation and lysosomal-related autophagy.GSEA enrichment analysis showed that the differentially expressed proteins in NETs and Control groups are related to P53 pathway,PI3K-δ/Akt/mTor pathway,MYC-related targets,inflammation,apoptosis,mTorc1 pathway,and erythromycin regulates inflammation caused by NETs It is related to TNF-α/NF-κB,inflammation,apoptosis,mTorc1 pathway,PI3K-δ/Akt/mTor pathway,IL-6/JAK/STAT3 pathway.The changes in the mTorc1 pathway,PI3K-δ/Akt/mTor pathway,IL-6/JAK/STAT3,and P53 pathway caused by NETs by erythromycin are consistent with the results of transcriptomics.2.Co-IP combined with mass spectrometry analysis showed that YWHAZ,HSPD1,ALB,PKM,HSPA8,EEF2,HSP90AA1,HSPA5,EEF1A1,RPSA,ANXA2,ACTB,TPI1,KRT19,ENO1,ACTA2,HSP90AB1,ATP5 B,KRT8,HSPA9 protein and HDAC2 protein had a closely relationship.3.Combined with the results of Co-IP and proteomics,it shows that differentially expressed proteins such as RPS8,RPS16,PA2G4,LYZ,CTSZ,RPL24,HADH etc form protein complexes with HDAC2.Conclusions: 1.In U937 cells stimulated by NETs,KEGG enrichment analysis of differentially expressed proteins showed that the inflammatory response caused by NETs may be related to oxidative stress and lysosomal autophagy-related pathways.Erythromycin can inhibit oxidative stress and Autophagy inhibits the inflammatory response caused by NETs.Combining the enrichment results of GSEA,KEGG,and GO with different proteins,combined with the results of transcriptomics analysis: In U937 cells stimulated by NETs,glucocorticoid resistance caused by NETs is associated with PI3K-δ/Akt/mTor pathway,inflammation,and apoptosis.The mTorc1 pathway is highly correlated.Erythromycin inhibits the glucocorticoid resistance of monocytes caused by NETs by inhibiting TNF-α/NF-k B,inflammation,apoptosis,mTorc1 pathway,PI3K-δ/Akt/mTor pathway.2.Proteins YWHAZ,HSPD1,ALB,PKM,HSPA8,EEF2,HSP90AA1,HSPA5,EEF1A1,RPSA,ANXA2,ACTB,TPI1,KRT19,ENO1,ACTA2,HSP90AB1,ATP5 B,KRT8,HSPA9 are involved in regulating the expression of HDAC2 in U937 cells.3.Differentially expressed proteins such as RPS8,RPS16,PA2G4,LYZ,CTSZ,RPL24,HADH etc form protein complexes with HDAC2,which proves that the differential protein is related to HDAC2-related glucocorticoid resistance,and it may be one of the intermediate targets in the biological process of erythromycin inhibits NETs induced glucocorticoid resistance.Part III Erythromycin attenuates monocyte glucocorticoid resistance induced by NETs stimulation via inhibiting PI3K-δ/Akt pathwayObjective: In order to further verify the mechanism of EM regulated glucocorticoid resistance and the omics results,RT-PCR and WB(Westernblot,WB)were used in the peripheral blood mononuclear cells of patients and U937 cells to verify the targets of glucocorticoid resistance caused by NETs and inflammation related with glucocorticoid resistance.Methods: 1.Clinical sample validation: experimental grouping: 1.Healthy group(control),2.Healthy smoking group(smoke),3.COPD group(COPD).Neutrophils and mononuclear cells were extracted from peripheral blood.Nets were induced from neutrophils of each group by CSE,and then mononuclear cells of each group were stimulated by Nets.WB was used to detect the protein expression of HDAC2,GR and Akt in monocytes.2.U937 cell verification: cell grouping: 1.Control group(control);2.Nets group(NETs): U937 cells were stimulated by nets at the concentration of 50 ng / ml for 24 hours;3.Erythromycin treatment group(EM + NETs: U937 cells were stimulated by Nets at the concentration of 50 ng / ml for 24 hours after incubation with erythromycin at the concentration of 1 ug / ml for 24 hours;4.Inhibitor group: PI3K-δ inhibitor ic87114 group(ic87114): U937 cells were stimulated by nets at the concentration of 50 ng / ml for 24 hours;4 RT1 inhibitor Nam group(NAM): 20 mmol / L for 24 hours,MAPK inhibitor sp600125 group(sp600125): 10 mmol / L for 24 hours.At the end of the modeling period,2 ’,7’-dichlorofluorescin diacetate(DCFH-DA,10 mm)was used as a fluorescent probe to observe the ROS release level of each group.The supernatant was collected and the level of IL-8 was detected by ELISA.The total protein was extracted for WB(Western Blot,WB)verification.The m RNA was extracted and the target gene was detected by RT-PCR.Results: 1.Nets stimulation in COPD patients can increase the expression of Akt and decrease the expression of HDAC2 and GR.2.Nets stimulation can increase ROS release in U937 cells,and erythromycin can improve ROS release induced by Nets.3.Nets stimulation can activate multiple Akt related pathways and targets,such as PI3 K / Akt / mTor,AP-1,NLRP3,MAPK,IKK / NF-κ B,SIRT1 / PPAR γ / PGC-1α The results showed that HDAC2 and GR activities decreased,GR phosphorylation decreased,IL-8 release increased and inflammatory reaction expanded.Erythromycin can reverse the changes of Akt related target activity induced by Nets stimulation,improve HDAC2,GR activity and GR phosphorylation level.It can also reduce the release of IL-8 and inhibit the inflammatory reaction.Conclusions: 1.Nets can induce oxidative stress and promote the release of ROS in U937 cells,which may be one of the mechanisms of nets activating inflammation related genes.Erythromycin can inhibit the release of ROS induced by nets,which is considered to be the effect of antioxidant stress of erythromycin.2.The stimulation of Nets leads to the changes of Akt related pathways and targets,and triggers inflammatory response,which eventually leads to glucocorticoid resistance.Akt is a key target of Nets related glucocorticoid resistance.Erythromycin improve glucocorticoid resistance induced by nets by inhibiting the activation of Akt and acting on related targets.